XRD and NMR based methods account for the majority of the high-resolution protein structures to date (Grey, J.; Thompson, D. H. Curr. Op. Drug Disc. 2010, 5, 1039). These methods, however, are of limited utility for structure determination of integral membrane proteins (IMP) and multiprotein complexes. Cryogenic electron microscopy (cryo-EM) has become an increasingly powerful method for determining the three-dimensional structures of proteins and their assemblies via Single Particle Analysis (SPA) (Cheng, et al. Ann. Rev. Biochem. 2009, 78, 723). Unlike other electron microscopy techniques, cryogenic electron microscopy (cryo-EM) does not require staining or embedding in non-physiological media. Recently, the technique has been used to determine protein structural information at 4.0 Å resolution (Cong et al. P Natl Acad Sci USA 2010, 107, 4967).
A key challenge for protein structural determination via cryogenic electron microscopy (cryo-EM) is that the method requires the collection of more than 30,000 images to enable image reconstruction at resolutions below ˜10 Å. Since protein concentrations for Single Particle Analysis (SPA) are typically in the μg-mg/mL range, data collection is a laborious and time-consuming process because there are typically only a few particles in the field of view at the magnification required to image the nano-scale targets. Thus, there is an ongoing need for the development of methods that increase the probability of finding the target particles in a vitrified sample to accelerate Single Particle Analysis (SPA) by cryogenic electron microscopy (cryo-EM).